Our long-term goal is to understand the mechanism(s) by which the hormone melatonin, through activation of its receptors (MT1 and MT2), regulates circadian, visual and neuroendocrine function. The overall goal of this proposal is to identify the molecular determinants and mechanism(s) by which melatonin differentially regulates hMT1 and hMT2 melatonin receptors and affects signal transduction in the mammalian central nervous system. Our hypothesis is that in the native suprachiasmatic nucleus (SCN) physiological concentrations of melatonin rapidly internalize and desensitize the MT2, but not the MT1 melatonin receptor altering the MT2-receptor mediated phase shift o f circadian rhythms. These studies will use state of the art molecular, biochemical and functional measurements in mammalian cells expressing recombinant hMT1 and hMT2 melatonin receptors and in native systems (SCN slice, SCN2.2 cells) to assess the mechanism(s) of melatonin induced receptor desensitization, internalization and cellular trafficking. Specific goals are: a) to identify the molecular determinants of MT1 and MT2 melatonin receptors involved in ligand induced internalization and desensitization and scaffolding proteins necessary for membrane residency; b) to determine homodimer and heterodimer formation in heterologous and native receptor systems; and c) to assess the functional sensitivity of MT2 receptor to mediate phase shift in the rat SCN slice and to stimulate protein kinase C (PKC) in immortalized SCN2.2 cells in culture upon exposure to physiological concentrations of melatonin mimicking the nocturnal surge. Understanding how MT1 and MT2 melatonin receptors are regulated by physiological concentrations of melatonin will increase our understanding of temporally regulated signal transduction events and identify potential targets for drug discovery. Specifically, these studies will shed light into the role of the circadian release of of melatonin in modulating functional responses in native target tissues and will be relevant to understand disturbances of circadian rhythms due to alterations in melatonin production at dusk and dawn. Furthermore, this research will increase our ability to design treatments aimed to alleviate symptoms associate with seasonal affective disorders.